Rapid diagnostic tests all employ various principles of separation and detection of analytes on membranes. However, the vast majority of these tests use colorimetric labels with a fixed amount of optically active substances such as colloidal gold, non-metal colloids, pigment particles/polymers or liposomes loaded with dyes. Test results are usually read by visual analysis of color intensity in diagnostic zones. The use of simple portable scanners/densitometers was also implemented into diagnostic practices to reduce operator mistakes in data interpretation or to integrate point-of-care (POC) testing into database system. However, these detection methods have inherent limitations in sensitivity related to absorptive characteristics of these detector labels. The analytical sensitivity of rapid tests utilizing colorimetric labels is significantly lower than the sensitivity of plate enzyme-linked immunoassay (ELISA), which is currently the major method of clinical diagnostics widely applied to screenings or confirmatory tests for diagnosis of diseases or health conditions.
The emergence of new diagnostic markers requires more sensitive detection systems. Numerous methods were developed to increase the analytical sensitivity. Among them were methods utilizing various fluorescent labels, super-paramagnetic labels, and chemiluminescent approaches. However, most of these methods required sophisticated equipment for reading, such as fluorimeters or magnetic readers, not readily compatible with POC applications of these tests or accessible in low resource settings. The availability of stable colorimetric detection systems for rapid tests providing analytical sensitivity attained at clinical laboratories should significantly extend the area of application of rapid tests for cost-efficient diagnostics and other analytical applications.
Peroxidase, in particular horseradish peroxidase (HRP), is one of the most important enzymatic labels for detection of analytes by various methods, such as microplate enzyme immunoassay (EIA), Western blot, dot-blot, immunohistochemistry and electrobiosensors. Low cost, high activity, stability, compatibility with various biological matrices and the availability of very stable forms of commercial substrates all make this enzyme a very popular label for diagnostic tests and various bioanalytical applications.
Direct detection of peroxidative activity of certain diagnostic markers and research analytes is also an important application for peroxidase substrate systems. One example is detection of peroxidative activity of human hemoglobin as a marker for color cancer. Emerging markers with peroxidative activity, such as glutathione peroxidase and myeloperoxidase, are considered important prognostic markers for myocardium infarction and coronary artery disease. Many other peroxidases such as microperoxidases, eosinophile peroxidases, uterine peroxidases, lactoperoxidases, salivary peroxidases, thyroid peroxidases, prostaglandin H1/2 synthase and enzymes of oxidative stress (superoxide dismutase), are potential markers for various metabolic dysfunctions.
Peroxidase substrates, which are suitable for colorimetric, fluorescent and chemiluminescent analysis are commercially available (e.g. from Molecular Probes/Invitrogen, Pierce and AnaSpec). The broad spectrum of substrates available for immunohistochemistry allows for multicolor labeling. The most popular substrates for tests performed in microplates or tubes are TMB (3,3′,5,5′-Tetramethyl-benzidine), DAB (3,3′-diaminodbenzidine), 1-Chloro-2-naphtol (CLN), diaminobenzidine, and ABTS (2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt). U.S. Pat. No. 6,960,323 also describes various urea derivatives, which may be used as a peroxidase substrate.
The detection of a peroxidase labels in EIA tests is based on the oxidation of a substrate with peroxidase in the presence of a peroxide (e.g. hydrogen peroxide). This reaction generates soluble reaction products. Precipitating substrates are commonly used for the detection of a peroxidase label, which is bound to a membrane (Western blot, dot-blot, dip-strip tests). When a peroxidase substrate is reacted with a peroxidase in the presence of a peroxide, an insoluble colored reaction product is produced, which precipitates onto the membrane where the peroxidase is captured. Examples of this type of substrate include TMB, 4-chloro-1-naphtol and DAB. Methods which use combinations of two precipitating peroxidase substrates (CN/DAB, TMB/CLN) and enhancers of dye precipitation, such as metal ions (e.g. Ni and Co), have also been described.
However, TMB precipitating substrates do have some shortcoming. For example, the adsorption of oxidized TMB polymers on membranes depends on the type of molecule immobilized in the zones, which capture the enzyme label. In some cases, due to the high density of the reagents, the TMB polymers fail to stick efficiently. Moreover, high concentrations of captured enzyme labels can result in the formation of excessive precipitating reaction products can result in diffusion outside of the zone. It is also very common to see high background staining due to non-specific adsorption of the enzyme onto the membrane in areas outside of the diagnostic zones. Dyes formed by TMB polymers as well as with other precipitating HRP substrates (1-Chloro-2-naphtol, diaminobenzidine) are light sensitive and quickly fade even without being exposed to direct light. All of these drawbacks limit the applicability of TMB substrate systems for quantitative densitometric analysis. Also, TMB substrate can not be prepared in a dry form, which could be quickly reconstituted with an aqueous buffer.
HRP substrates producing light insensitive indamine type dyes as a complex of MBTH (3-methyl-2-benzothiazolinone hydrazone) with dienophile or an aromatic nucleophile is described in U.S. Pat. No. 5,432,285. Another HRP precipitating substrate system utilizes a combination of two reagents: 4-CN and MBTH or substituted p-phenylenediamine (Conyers, S M, Kidwell D A. Anal Biochem, 1991, 192: 207-211).
Oxidative coupling of 3-(dimethyl aniline)benzoic acid (hydrogen donor) with MBTH (3-methyl-2-benzothiazolinone hydrazone) (oxidizable coupling reagent) was described in Ngo et al (Anal Biochem, 1980, 105, 389-370). This method is similar to a method, which uses 4-aminoantipyrine as an oxidative coupler with a phenolic compound and aniline (Trinder, P., Ann. Clin. Biochem 6, 24-25 (1969)). In these methods, a hydrogen peroxide-oxidized form of HRP reacts first with an oxidizable compound (coupler), which then reacts quickly with a second compound (hydrogen donor) in a reaction of electrophilic substitution. The reaction proceeds without the participation of HRP to form strong chromophores, which are usually soluble compounds. This method was adapted for oxidative coupling of 3-(dimethyl aniline)benzoic acid with 3-methyl-2-benzothiazolinone hydrazone as a soluble substrate system for plate EIA tests (Georghegan et al, J Immunol Methods, 1983, 60, 61-68). These chromogenic systems produce intense colors that are relatively stable. However, the hydrogen donors used in these systems are unstable in solution. In addition, the sensitivity of this type of substrate for EIA assays is lower than the sensitivity of the best TMB HRP substrate.
Another method for detecting peroxidases, based on a two component dye system, was developed using leucodyes, which produce colors on membranes in the presence of an electron transfer reagent such as 4-hydroxyacetanilide (U.S. Pat. No. 5,024,935) and 3-aminopyrazolo heterocyclic derivatives in combination with aniline derivatives (U.S. Pat. No. 5,457,200).
A number of U.S. patents describe oxidative coupling of 4-aminoantipyrine or sulfoMBTH with various aniline derivatives including fluorinated derivatives (See e.g. U.S. Pat. Nos. 4,845,030 and 4,912,258).
Perhaps the most developed rapid diagnostics utilizing a two-component oxidative coupling dye system are enzymatic tests for the detection of low molecular weight substances such as glucose, cholesterol, uric acid, choline, alcohol, lactate, ascorbic acid, acetyl-CoA and certain drugs. In these systems, hydrogen peroxide is generated when the low molecular weight substance is oxidized with an appropriate enzyme (cholesterol oxidase, glucose oxidase, lactate oxidase, uricase, choline oxidase, ascorbate oxidase guanase, etc.). The hydrogen peroxide produced is then consumed by HRP, which uses a two component dye system to produce color. The amount of hydrogen peroxide produced correlates with the amount of the particular low molecular weight substance and the amount of dye generated by the HRP is based on the enzymatic conversion. The reagents for this substrate system may be distributed throughout the length of a diagnostic strip, so that the length of the colored zone on the diagnostic strip correlates quantitatively with the amount of test analyte in the sample.
Numerous patents describe two-component dye systems utilizing various hydrogen donor/coupler combinations for analyte detection in a strip format. The result may be visually read or read using a reflectance spectrophotomer or photometer (U.S. Pat. Nos. 6,858,401; 6,635,439; 6,531,322; 6,218,571; 5,972,294; 5,922,530 and 5,824,491). These tests typically require a liquid sample. Substrates that do not contain hydrogen peroxide are stable if prepared as a slowly soluble dry film or non-porous materials.
Several patents describe the use of hydrogen donors covalently immobilized onto a solid phase. For example, the quinoline type hydrogen donor, 8-(4-amino-1-methylbutylamino)-6-methoxyquinoline (primaquine) has been coupled to carbonyldiimidazole activated cellulosic membrane (U.S. Pat. No. 5,556,743) and used as part of a strip test for detecting cholesterol. A similar approach has been described for the immobilization of an aniline derivative with amino groups on CDI (carbonyldiimidazole) activated cellulose membrane (U.S. Pat. No. 5,155,025). Aniline derivatives with primary amino groups such as N-alkyl 3-oxyanilines were synthesized as insoluble polyvinyl alcohol polymer conjugates (U.S. Pat. No. 5,409,780). Such insoluble polymers were applied as plastic films and used as a rapid test for testing glucose in blood or urine.
Various patents also describe various oxidative couplers (e.g. MBTH, 4-aminoantipyrine, analogs of 4-aminoantipyrine, a modified MBTH with increased solubility, which contains a carboxyl group (U.S. Pat. No. 5,710,012); a salt of 6-carboxy-3-methylbenzenothiazolene hydrazone hydrate; MBTH derivatives that are sulfonated on the ring (U.S. Pat. No. 6,242,207); 3-methyl-6(sulfonate salt)-benzothiazolinone-(2)-hydrazone) or an N-sulfonyl benzensulfonate derivative (U.S. Pat. No. 5,992,530); meta[3-methyl 2-benzothiazolinone hydrazone]N-sulfonyl benzenesulfonate monosodium) or as dinitrobenzene derivatives (U.S. Pat. No. 4,962,040) and 2-hydrazono-4,6,-dinitrobenzthiazolone). Other combinations of hydrogen donor/couplers for the determination of peroxidatively active catalysts such as amino benzidine type hydrogen donors and substituted phenol type couplers are described in U.S. Pat. No. 5,532,138.
The vast majority of currently available membrane-based immunodiagnostic tests that utilize peroxidase and another enzyme label, use principles of vertical filtration (flow through), dip-sticks and other formats, which rely on passive diffusion. The basic components of flow-through rapid diagnostic kits are liquid reagents, HRP conjugates, HRP substrates and a wash buffer. A precipitating substrate system (usually a TMB based substrate) is typically used to detect the peroxidase activity of a bound enzyme.
Lateral flow (LF) rapid tests with enzyme detector labels, which generate dyes, currently represent a very small segment of the diagnostic market. The advantages of using highly sensitive enzyme labels are difficult to obtain in simple LF rapid tests, which utilize only dry components. Enzyme labels require an additional component, the substrate, and in most cases require a more efficient washing step, which imposes additional challenges and costs.
Certain patents describe lateral flow devices, which utilize liquid or dry HRP conjugates and liquid TMB precipitating substrates integrated into diagnostic cassettes as a sealed container together with a container of wash buffer. These containers can be perforated to initiate the flow of wash buffer and subsequent delivery of substrate into the diagnostic zones on a fast flow porous polyethylene matrix (U.S. Pat. Nos. 7,442,557; 6,436,722 and 5,726,010). Positive results can typically be measured visually after about 8-10 minutes.
Certain patents describe LF strips with dual paths, one for the delivery of any enzyme labeled analyte, and the other for the delivery of reconstituted substrates for alkaline phosphatase or beta-galactosidase based systems) with appropriate washing separating these two processes (U.S. Pat. No. 6,706,539). Other patents describe devices, which contain zones of immobilized substrates. These zones generate color when an enzyme enters by diffusion through a permeable barrier (U.S. Pat. No. 4,806,312). Lipid vesicles (e.g. liposomes) loaded with enzyme substrates may also be captured into a test zone. In the presence of a specific analyte and enzyme label, the vesicles can release the enzyme substrate into the test zones with the aid of a phospholipase, which has been incorporated into a capture zone.
Commercially available dip-strip tests utilize porous or non-porous materials to immobilize capture reagents, liquid enzyme conjugates and substrate reagents. The whole strip or a working portion may be incubated in a series of solutions (e.g. sample, conjugate, washing solution and substrate).
The use of HRP as a label for conventional LF tests faces other technical challenges. Among the most critical is the absence of stable dry forms of peroxidase substrates containing a peroxide compound. Also many peroxidase substrates, such as TMB, are not very soluble. And although special additives may be used to maintain TMB in solution, these mixtures cannot be prepared in an appropriate dry form suitable for quick solubilization (See U.S. Pat. No. 5,910,423). Other peroxidase substrates, which produce precipitating products require organic solvents to maintain their solubility, some of which are carcinogenic. Glucose oxidase co-immobilized with capture antibodies on membranes can produce hydrogen peroxide for HRP chromogenic reactions in the presence of glucose. This approach was applied for quantitative analysis of low-molecular weight analytes in competitive tests where the length of the colored zone on the strip is a measure of the analyte concentration (Li et al. Analyt Biochem, 1987, 166, 276-83, Zuk et al. Clin Chem, 1985, 7, 1144-50). However, this approach has limited analytical sensitivity.
All of the currently available substrate systems for peroxidase produce a monochromic color, due to the precipitation of a common dye polymer. The internal control zones and test zones on these membranes typically only generate a color in the violet-blue range.
Accordingly, there is a need for more sensitive peroxidase substrate based rapid tests, which employ stable dry reagents.